Multiplexed Single‐Particle Imaging Enabled by Modulation of Er ³⁺ Energy‐Level Populations

Single-particle tracking (SPT) offers critical insights into nanoscale molecular dynamics, but is limited by short tracking durations due to irreversible photobleaching and the technical complexity of multicolor imaging. Here, a non-photobleaching ratiometric imaging strategy is developed based on lanthanide-doped upconversion nanoparticles (UCNPs), exploiting their intrinsic, tunable red-to-green (R/G) emission ratios for multiplexed SPT. Single-particle characterization reveals over 10-fold tunability. Mechanistic investigations show that this ratiometric behavior is governed by Yb³⁺ excitation density, which modulates the energy distribution within energy levels of Er³⁺ ions. Specifically, high Yb³⁺ excitation densities enhance three-photon transitions, favoring red emission, while lower densities promote two-photon upconversion and green emission. Both processes proceed through a shared energy level of ²H_11/2, leading to competitive energy transfer dynamics. Based on this competition mechanism, a quantitative relationship is further established between UCNPs structure and the resulting R/G emission ratio, allowing reliable prediction of spectral output across different designs. Leveraging this tunable ratiometric principle, simultaneous five-color single-particle imaging is demonstrated with a misidentification rate below 5%. This strategy is further applied to visualize receptor-mediated endocytosis in live cells. This work highlights the advantages of upconversion luminescence-based R/G ratio discrimination for long-term, multicolor SPT, offering a simple and reliable tool for probing complex biological processes.